CHAPTER FOUR ADSORPTION OF STRAINS OF RHIZOBIUM
JAPONICUM WITH DIFFERENTIAL NODULATING ABILITY TO
ROOTS OF SOYBEAN ISOLINES THAT DIFFER AT THE Ril
LOCUS............................ ................ .. 57

rJlrJl-plants. The biological significance of this latter
finding is not apparent in light of Elkan's (1962) earlier

data on root colonization, which certainly suggests no basic

growth inhibition of rhizobia. Elkan (1961) suggested that

the rijilr-soybean produced a nodulation-inhibiting

excretion capable of a highly significant reduction of

nodulation on the Rjl-genotype. The amount of nitrate added

to culture medium for container-grown plants, however, was

sufficient to have a potential effect on nodule number.

Eskew and Schrader (1977) reexamined the putative

nodulation-inhibiting excretion from rjl-soybean using

modifications of Elkan's (1961) experimental design. They

found no statistically significant reduction in nodule

number due to co-cultivation of nodulating plants with rjl-

isolines, but a strong inhibitory effect of nitrate was

noted.

Hubbell and Elkan (1967a) compared thephysiological

characteristics of strains of R. japonicum with differential

abilities to nodulate isogenic lines of soybean differing at

the Rjl locus. High measurable indoleacetic acid formation,

low indoleacetic acid destruction, formation of large

amounts of capsular material, failure to metabolize nitrate,

and failure to reduce tr.iphenyl tetrazolium chloride were

properties associated with ability to nodulate both normal

and mutant soybean. Stains with the opposite properties

generally were able to nodulate only the normal soybean. A

mode of infection could not be educed from correlations

between physiological characteristics and nodulation

phenotype.

Devine and Weber (1977) observed that many R. japonicum

strains capable of overcoming ril-conditioned resistance to

nodulation produced a previously reported soybean foliar

chlorosis (Erdman et al. 1956, Johnson and Means 1960). The

chlorosis symptoms were ascribed to the formation of

rhizobitoxine, 2-amino-3-hydroxypropoxyvinylglycine by the

bacteria (Owens and Wright 1965, Owens 1969, Giovanelli et

al. 1971, Owens et al. 1972). Devine and Weber (1977)

suggested that the production of rhizobitoxine might enable

the infection of rilril-soybean by overcoming strains. This

was examined indirectly by Devine and Breithaupt (1980b),

who tested the effect of three temperature regimes on

nodulation and chlorosis. The effects were opposite, in

that the chlorosis symptoms were greatest at the highest

temperature (32 C), but the most nodules were formed at the

low and intermediate temperatures (21 C and 27 C). No

evidence was found for a diffusible compound capable of

endowing the rjl-incompatible strains with ability to

nodulate rjillj-soybean (Devine et al. 1981). The ethoxy

analog of rhizobitoxine, when added to bacteria and used to

inoculate soybeans in Leonard jars, did not modify the

nodulating ability of strains of R. japonicum on rjlrjl-

soybean (Devine and Breithaupt 1980a). Devine (1984a)

concludes from these data that rhizobitoxine probably has no

enabling role in infection. Devine suggests that the

rhizobitoxine is only correlated with the rJilr1-overcoming

strains due to fixation of the separate genetic factors in

the same population by "random drift."

Devine et al. (1980) determined that the _ll1-

resistance was not a basic incompatibility with the

nonnodulating stains. When the strains capable of

nodulation and those which were not were mixed and used as

inoculum, 32% of the resulting nodules on rllr_1i-soybean

contained both strains, 36% contained only the usually

nonnodulating strain, and 32% contained only the usually

nodulating stain.

The mode of infection of rilj l-soybean has not been

determined. Nutman (1981) notes that the rr phenotype of

red clover conditions inability of the plant to form

infection threads; and, since the soybean resistance to

nodulation is likewise a recessive trait, it seems likely to

condition a similar block early in infection. Devine

(1984a) notes that no nodules or nodule-like proliferations

are formed on roots inoculated with incompatible strains,

and likewise suggests that the block is early in infection.

Tanner and Anderson (1963) examined soybean roots for

infection in root hairs but unfortunately were unable to

find infection threads in either the 1ilrjl-soybean or the

normally nodulating line.

Perspective

The challenge remains to find the point at which the

ril-plant blocks infection and to elucidate the pathway of

-34-

infection for those strains which can overcome the rilrjl-

resistance to nodulation. These problems are the focus for

the studies reported in this dissertation. The function of

temperature on nodulation number and pattern was studied to

find the conditions under which infections were most likely

to be observed in the rjillj-soybean. The hypothesis that

bacterial adsorption has a role in determining differential

nodulation ablilty of strains between normally nodulating

and restrictive lines of soybean was tested. The plasmid

content of strains was determined and attempts were made to

alter the genetic complement of strains, in search of clues

to the genetic basis for infection. Finally, roots were

examined using light and scanning electron microscopy to

determine the phenotype of infection at the cellular level.

CHAPTER THREE
EFFECT OF TEMPERATURE ON NODULATION OF SOYBEAN ISOLINES
DIFFERING AT THE Ril LOCUS

Introduction

The nodulation restrictive genotype of soybean, rjilrl,

identified as a spontaneous mutant in a soybean breeding

program, was reported by Williams and Lynch in 1954.

Initial work determined that one genetic locus is involved

in conditioning the restrictive phenotype and that the

homozygous recessive genotype is required for expression of

the trait. The alleles originally were named no and NO for

nonnodulating, but since have been redesignated Kil and Rj1

to conform to currently accepted terminology for soybean

genetics (Caldwell 1966).

It once was believed that the nodulation restrictive

plants are unable to be nodulated (Williams and Lynch 1954,

Caldwell 1966). Now it is clear that although most strains

of Rhizobium japonicum are unable to nodulate these plants,

several strains produce a small number of nodules on plants

grown in hydroponic culture (Clark 1957). These strains are

called the "overcoming" strains because they overcome the

plant resistance. Devine and Breithaupt (1980b) reported a

temperature effect on nodulation of the soybean cultivar

Clark and its nodulation-restrictive isoline Clark-ril by

-35-

two overcoming strains. The trends in nodulation response

of both isolines to temperature are similar. The two

bacterial strains have different temperature optima for

nodulation with the greatest number of nodules per plant

formed at 27 C and 21 C, respectively, for the two strains.

Fewer nodules were formed at 32 C for both strains (Devine

and Breithaupt 1980b).

Bhuvaneswari and colleagues (Bhuvaneswari 1981,

Bhuvaneswari et al. 1980, 1981) developed a model for

nodulation of soybean. The model predicts that most nodules

will be clustered near the point that represents the

position of the root tip at the time of inoculation. The

model is supported by a correlation between nodule

distribution and the position of areas that had immature

root hairs or had not yet developed root hairs at the time

of inoculation. This developmental model of nodulation is

extended by the observations of Pueppke (1983) and Calvert

et al. (1984), who demonstrated that the formation of

infection threads is the developmentally restricted event in

soybean and two other legumes. In accordance with this

model, no nodules are expected to form above the zone of

developing root hairs on the primary root (Bhuvaneswari et

al. 1980).

The objectives of my study were to i. find the

temperature optima for overcoming strains, ii. test the

appropriateness to rjflrl-soybean of the Bhuvaneswari model

of transient susceptibility of root cells to infection

leading to nodulation, and iii. extend the study (Devine and

-37-

Breithaupt 1980b) of the effect of temperature on nodulation

of Clark and Clark-rji isolines to additional overcoming

strains.

Materials and Methods

The bacteria all were obtained from the U. S.

Department of Agriculture, Nitrogen Fixation and Soybean

Genetics Laboratory, Beltsville, MD, courtesy of H. H.

Keyser, D. F. Weber, and R. Griffin. All bacteria are

USDA strains of R. aponicum. The overcoming strains used

were 61, 84, 94, and 119. The nonovercoming strain 110 was

used as a control in all experiments. The bacteria were

maintained at 4 C on yeast extract-mannitol agar slants

(Vincent 1970).

Seeds of Glycine max (L.) Merr. cultivar Clark-L1

(RjilRj) and the nodulation restrictive isoline of Clark-Ll,

L63-1889 (rjlil), were obtained from R. L. Bernard, USDA

Regional Soybean Laboratory, University of Illinois, Urbana,

and D. A. Phillips, Agronomy and Range Science Department,

University of California, Davis. The isolines are

designated Clark and Clark-ril according to the nomenclature

of Devine and Breithaupt (1980b). Seeds were surface

disinfested by soaking in 50% ethanol for 2 min with

agitation, rinsing in deionized water, and then shaking in

0.5% aqueous sodium hypochlorite for 2 min. Seeds were

washed for 20 min in running deionized water and were

germinated in the dark on water agar plates for 4 to 5 d at

22 C, 27 C, or 32 C, depending on the temperature to be used

for nodulation experiments.

Inoculum was produced from 50 ml log-phase cultures

grown in liquid gluconate-mannitol medium (Bhuvaneswari et

al. 1977) at 28 C with rotory shaking at 120 rpm. Bacterial

suspensions were centrifuged at 7500 x g for 10 min and the

bacteria were resuspended in sterile nitrogen-free Jensen's

plant-growth solution (Vincent 1970). Cell concentration

was adjusted turbidimetrically to 5 x 108 cells/ml.

Seedlings with roots approximately 4 cm long were inoculated

by dipping the roots for 10 min in the bacterial suspension.

Inoculated seedlings were placed, 2 per pouch, in

autbclaved plastic growth pouches (Northrup King Seed Co,

Minneapolis, MN) containing 15 ml of Jensen's solution. The

surface of the growth pouch was marked at the location of

the primary root tip of each plant (Bhuvaneswari et al.

1980b). This mark was designated the root tip mark (RTM).

Plants were grown for 30 d at continuous temperatures

of 22, 27, or 32 C with a 12 hr light/dark cycle in a

Conviron E-15 growth chamber with 900 uE/m2/sec (400-700 nm)

irradiance at canopy height. Each temperature experiment

was repeated 3 times with 6 plants of Clark and 10 plants of

Clark-ril tested for each R. japonicum strain in each

experiment. Appropriate control plants sham-inoculated with

sterile Jensen's solution were included in each experiment.

Plants were watered as needed with deionized water.

The distance (to the nearest mm) from the RTM to the

root crown and from the RTM to each nodule on the primary

-39-

root was measured on each plant at harvest. The number of

nodules on secondary roots was counted. Data were analyzed

by analysis of variance using the general linear models

(GLM) procedure of the Statistical Analysis System (SAS

Institute Inc., Cary, NC). Computing was done utilizing the

facilities of the Northeast Regional Data Center of the

State University System of Florida.

Results

The mean number of nodules per plant on Clark ranged

from 2 to 8 at 32 C, up to 15 to 19 at 22 C (Figure 3.1,

Table 3.1). On Clark-ril inoculated with overcoming

strains, the mean number of nodules ranged from 0 to about

0.2 nodules per plant at 32 C, to from 0.7 to 2.2 nodules

per plant at 22 C (Table 3.2, Figure 3.1). Nodules were not

formed on Clark-rjl inoculated with the nonovercoming strain

110 or on plants sham-inoculated with plant-growth medium.

Analysis of variance was conducted using the number of

nodules per plant as the dependent variable. In a

preliminary test, the responses of Clark and Clark-ril were

demonstrated to be significantly different. All subsequent

analysis thus was conducted separately for the two genotypes

to avoid heterogeneity of variance. Temperature

significantly (p < 0.01) influenced nodule number for each

plant genotype. F-values for strain, replication, and the

interactions of each of the independent variables were

Figure 3.1. The mean number of nodules formed per
plant at three temperatures with five strains of
Rhizobium japonicuTr Plants were dip inoculated in
suspensions (5 x 10 cells/ml) of one of the strains
indicated, placed in plastic growth pouches, and grown
at constant temperature for 30 d. For each strain at
each temperature the experiment was replicated three
times with six plants per treatment for Clark and ten
plants per treatment for Clark-rjI. Treatments with
Clark are indicated by the solid line, Clark-rjI with
the broken line. 0= strain 61, U = strain 84, 6 =
strain 94, 0= strain 110, and A = strain 119.

20-

19-

18-

17-

16-

15- 0

14-

13 -

12 -

'11 -

-10- a

S 8 --

a- a,
5-

14-

3-

2-

22 27 32
TEMPERATURE ICI

Table 3.1. The effect of temperature on the nodulation of
Clark soybean by Rhizobium japonicum

Temperature

Strain 22 C 27 C 32 C

15 6a

16 1 7

18 5

16 9

19 7

12 4

10 3

15 6

12 5

15- 6

8 3

7 3

2 2

7 3

6 3

a Mean number of nodules per plant for 3 replications with
6 plants per replication standard deviation.

a Strain 110 is a nonovercoming control. All others are
overcoming strains.

b Mean number per plant for 3 replications with 10 plants
per replication standard deviation.

-44-

not significant. A dramatic indication of the effect of

temperature on nodulation of Clark-irj is provided when the

data are expressed as the percentage of plants developing at

least one nodule per plant after inoculation with an

overcoming strain (Table 3.3). At each temperature a total

of 120 plants was inoculated with one of the four overcoming

strains; of those, 4% of the plants were nodulated at 32 C,

23% at 27 C and 44% at 22 C.

The ratio of primary to secondary nodules ranged from

1:0.6 to 1:0.9 on Clark. On Clark-rjI the ratios were 1:9

at both 32 C and 27 C, and 1:4 at 22 C.

Histograms were developed for each interaction of

bacterial strain x isoline for each temperature as described

for various other legume x microsymbiont combinations

(Bhuvaneswari 1981, Bhuvaneswari et al. 1980, 1981,

Halverson and Stacey 1984, Heron and Pueppke 1984). These

are shown in Figures 3.2 and 3.3. In most interactions with

overcoming strains, nodules formed well above what has been

considered the zone of infectibility, defined as that area

which has only emerging root hairs or no root hairs at the

time of inoculation. This type of anomolous nodulation is

seen at 22 C and 27 C for combinations of Clark with the

strains 61, 84, and 94, each an overcoming strain. A

pattern of nodulation similar to those described as fitting

the nodulation model of Bhuvaneswari (1981) is seen in the

combination of Clark with strain 110, a nonovercoming

strain, at 22 C and 27 C. From the nodule profiles

-45-

(Figure 3.2), it can be seen that the pattern of nodulation

at 32 C of Clark by all of the tested bacterial strains

produced flattened peak and a population of nodules

displaced downward with respect to the profiles observed at

22 C and 27 C. This downward displacement is clearly

evident when the data are expressed as the mean distance of

all primary root nodules from the RTM (Table 3.4). The

nodule profiles of the overcoming strains on Clark-ril

showed sparse nodulation down the length of the root from

just above the RTM.

Discussion

The soybean cultivar Clark and its isoline, Clark-ril,

were used by Devine and Breithaupt (1980b) to study the

effect of temperature on nodulation. They tested the two

overcoming strains USDA 61 (used in this study) and 76.

Strain 76 produced the most nodules on Clark-ril at 27 C

with few nodules formed at 21 C or 32 C. The combination of

Clark-rjl with strain 61 developed the most nodules at 21 C

(7.5), with 6.4 and 3.1 nodules at 27 C and 32 C,

respectively. In my study the slope of the regression of a

plot--number of nodules versus temperature--was similar to

that observed by Devine and Breithaupt (1980b), but the

absolute numbers of nodules per plant were lower (Figure

3.1). All of the overcoming strains that I tested responded

similarly to strain 61 in the previous study, except strain

84 which had slightly fewer nodules at 22 C than at 27 C.

-46-

Table 3.3. The percentage of Clark-rj1 soybean plants
nodulated by overcoming strains of Rhizobium japonicum
at three temperatures

Temperature

22 C 27 C 32 C

Strain pri sec anya pri sec any pri sec any

61 23 73 77

84 3 30 30

94 13 27 40

10 50 53

7 30 30

0 10 10

3 3 7

0 10 10

0 0 0

3 27 30

0 0 0 0 0 0

Totalb 11 39 44

4 23 23

1 3 4

a Percentage of 30 plants nodulated (3 replications x 10
plants) at the locations indicated. Pri = percentage of
plants with at least one nodule on the primary root.
Sec = percentage of plants with at least one nodule on
the secondary roots. Any = percentage of plants with
at least one nodule.

b Percentage of all plants in each temperature experiment
with at least one nodule (120 plants per temperature).

Figure 3.2. Frequency histogram of nodule distribution on the primary root of Clark
soybean. Plants were grown 30 d in plastic growth pouches at 22, 27, or 32 C after
dip inoculation with one of the five strains of Rhizobium japonicum indicated. Each
nodule on the primary root was measured to the nearest millimeter from the RTM (root
tip mark, placed on the growth pouch at the time of inoculation). The frequency
diagrams represent the plants from three replications at each temperature with six
plants per treatment.

STRAIN 61
22 C 27 C 32 C

STRAIN 84
22 C 27 C 32 C

40

20

RTM

20

40

60

80

100

120

140

STRAIN 94

STRAIN 94
22 C 27 C 32 C

-

r[

STRAIN I 10

22 C 27 C 32 C

-= ONE
NODULE

Figure 3.2 Continued

STRAIN 119

22 C 27 C 32 C

40

20

RTM

20

40

60

80

100

120

140

Figure 3.3. Frequency histogram of nodule distribution on the primary root of
Clark-rjl soybean. Plants were grown 30 d in plastic growth pouches at 22, 27, or
32 C after dip inoculation with one of the four strains of Rhizobium j~iponicum
indicated, or the control strain 110. Each nodule on the primary root was measured
to the nearest millimeter from the RTM (root tip mark, placed on the growth pouch at
the time of inoculation). The frequency diagrams represent the plants from three
replications at each temperature with ten plants per treatment. No nodules were
observed on plants inoculated with strain 110 (not shown) at any temperature.

-51-

3 I 3

-I
I0
-I

IC,

cm
10.
IC

CI

nc
u

I
.c-
IN

C.,
-I o
CM

0 0--0--0 0-0-0-0- -

Ca I- C,' o
= UC h DQ C

-52-

Table 3.4. Mean distance of primary root nodules on Clark
soybean from the root tip mark at time of inoculation

Temperature

Strain 22 C 27 C 32 C

16 2a

16 2

11 2

16 1

61 5

56 4

13 2

16 2

7 2

14 1

9 1

53 7

43 -+ 5

a Mean nodule distance in millimeters for 18 plants (3
replications x 6 plants) standard error of the mean.
Measurements for nodules above the RTM were given a
negative value.

bOnly one nodule produced on the primary root of a plant
in 3 replications of this treatment.

-53-

At 22 C only the combination of the nonovercoming strain 110

and Clark-ril failed to produce nodules on at least some of

the plants. At 27 C, Clark-rj1 failed to form nodules with

strain 119 as well as with strain 110, and at 32 C no

nodules were formed by strains 94 and 119, as well as 110.

The effect of temperature on nodulation of both Clark and

Clark-rj1 had the same trend (Figure 3.1), although the

number of nodules per plant was much different for the two

plant types. Nodulation of nonovercoming strain 110 on

Clark was affected by temperature in a manner similar to the

overcoming strains, but strain 110 did not nodulate the

Clark-ril isoline at any temperature.

Reports have been made on the effect of temperature on

numbers of soybean nodules per plant both in the greenhouse

and field, as well as under controlled conditions (Devine

and Breithaupt 1980b, Munevar and Wollum 1982, Weber and

Miller 1972), but this study is the first to examine the

effect of temperature on the pattern of nodulation. The

pattern of nodulation of strain 110 on Clark soybean at 27 C

and 22 C was similar to the pattern reported by others for

compatible interactions and follows that predicted by the

model of transient susceptibility to nodulation (Bhuvaneswari

1981, Bhuvaneswari et al. 1980, 1981). The general shape of

the profiles for the overcoming strains on Clark is similar

to strain 110 on Clark, suggesting that at least most of the

nodules that arise are the result of infections constrained

by developmental processes to areas of susceptibility, as

-54-

defined by the region of the root on which the root hairs

are immature or are not yet formed at the time of

inoculation. Overcoming strains 61, 84, and 94 produced some

nodules well above that region known to be infectible by the

model of infection. The presence of these nodules can be

explained in two ways. First, immature root hairs may exist

in this region and remain infectible after the surrounding

root hairs have matured. Alternatively, these overcoming

strains may have an additional infection sequence that is

not restricted to areas with developing root hairs. If the

first is true, one would expect that strain 110 and other

nonovercoming strains also would produce nodules in this

area, at least occasionally. Since nodules are produced in

this area only by overcoming strains, an alternative

infection mechanism is suggested.

The pattern of nodulation at 32 C of Clark soybean with

the five strains of R. japonicum gave a more flattened curve

which was displaced downward relative to those at the lower

temperatures. Nodulation generally was lower on the primary

root, and some nodules were very far below the RTM. The

pattern of nodulation obtained at this temperature looks

much like that reported (Halverson and Stacey 1984, Heron

and Pueppke 1984) for interactions with fewer nodules

relative to other interacti-ons tested in those studies.

Heron and Pueppke (1984) reported a similar pattern on the

soybean cultivar Vicoja inoculated with the fast growing R.

japonicum strain 191. Halverson and Stacey (1984) reported

that the delayed nodulating mutant strain HS111 produced a

-55-

similar scattered and downwardly displaced pattern on Essex

soybean as compared to the pattern obtained with strain 110.

An explanation for the striking similarity in the

nodulation pattern for all of these interactions is that the

they are merely diagnostic for any interaction in which the

initial number of successful infections is reduced and plant

regulation of additional nodulation is not triggered (Pierce

and Bauer 1983). That is, the similarities of the patterns

may be coincidental. But the surprising similarity of those

nodulation patterns to the ones in this study resulting from

restrictive temperature, brings up the question as to what

effect temperature might have on those interactions. The

soybean cultivar Vicoja was developed at the Universidade

Federal de Vicosa, Vicosa, Minas Gerais, Brazil,

specifically for local Brazilian conditions, including high

temperature. Thus the observed inefficiency of nodulation

is perhaps due to assay temperatures below those to which

Vicoja is adapted. Similarly, the possibility that HS111 is

a temperature sensitive mutant that has been tested only at

restrictive temperatures cannot be ruled out.

The nodulation of Clark-r.E is so sparse that

interpretation of the nodule profile is difficult. There is

certainly no clear peak in the histogram near the point

corresponding to the RTM. Whether such a peak would become

evident if much greater numbers of plants were examined is

uncertain, but seems unlikely; the pattern appears to be

scattered and random. Although these data are insufficient

-56-

to either validate or invalidate the application of the

model of Bhuvaneswari (1981) to Clark-riI, these data show

only limited correspondence with the expected pattern of

nodulation predicted by the Bhuvaneswari model. The

scattered and sparse nodulation and the reduced correlation

of nodulation with the region near the RTM are likely to

make the elucidation of early infection events in the

nodulation restrictive rj1 rj-soybean that much more

challenging.

CHAPTER FOUR
ADSORPTION OF STRAINS OF RHIZOBIUM JAPONICUM WITH
DIFFERENTIAL NODULATING ABILITY TO ROOTS OF SOYBEAN ISOLINES
THAT DIFFER AT THE Rji LOCUS

Figure 4.1. Adsorption of cells of Rhizobium japonicum
to soybean roots. The experiments were completed at 27
C. Each point represents the mean from five
experiments with four pairs of plants tested at each
time for each plant-strain combination at each
replication of the experiment. The roots of soybean
seedlings were incubated in bacterial suspensions (1 x
10 cells/ml) for the times indicated and rinsed
vigorously. The terminal 2 cm of the primary roots
were excised, ground and plated for determination of
colony forming units. O = Clark x strain 110, U =
Clark x strain 94, O = Clark-ril x strain 110, and
* = Clark-rji x strain 94.Bars represent the standard
error of the mean.

-68-

0 30 60 90
MINUTES

150

I-

t--oo
.100

" 50
I-

120

Figure 4.2. Adsorption of cells of Rhizobium japonicum
strain 94 to roots of Clark-rj1 soybean at two
temperatures. Each point represents the mean of four
pairs of plants tested at each time for each
temperature from each of five replications of the
experiment. The roots of seedlings were incubated in
bacterial suspensions (1 x 10 cells/ml) equilibrated
at 22 C or 27 C for the times indicated and rinsed
vigorously. The terminal 2 cm of the primary roots
were excised, ground and plated for determination of
colony forming units. V = 22 C, and 0 = 27 C (The 27
C curve is duplicated from Figure 3.2.). Bars
represent the standard error of the mean.

30 60 90
MINUTES

150

100

50

120

Table 4.1. Percentage of plants nodulated at 27 C and 22 C

Primary Secondary Any Root

Combination 27 C 22 C 27 C 22 C 27 C 22 C

Clark x 94

Clark x 110

Clark-rjl x 94

Clark-ril x 110

89a 100 89 100 100 100

89 100 50 89 100 100

0 13 10 27 10 40

0 0 0 0 0 0

a Data in table from Chapter Three. Percentage of plants
nodulated at each indicated location. Data are for all
plants from 3 replications at each temperature with 6
to 10 plants per replication.

Table 4.2. Mean number of nodules per plant at 27 C and 22 C

Primary Secondary Any Root

Combination 27 C 22 C 27 C 22 C 27 C 22 C

Clark x 94

Clark x 110

Clark-ril x 94

Clark-rjl x 110

8.8a 7.9

8.3 8.9

5.8 10.2 14.6 18.1

3.6 6.8 11.9 15.7

0 0.3 0.1 0.5 0.1 0.9

0 0 0 0 0 0

a Data in table from Chapter Three. Mean number of
nodules per plant on primary roots, secondary roots, or
on any root of the plant as indicated. Each value
represents the average for 3 replications at each
temperature with 6 to 10 plants per replication.

-73-

Table 4.3. Nodulation of plants inoculated with Rhizobium
japonicum under the conditions of the adsorption assay

a Seedlings were inoculated by dipping the roots in a
bacterial suspension containing approximately 104
cells/ml for 2 hr. The roots were rinsed vigorously.
Plants were grown for 20 d in plastic growth pouches
(see Materials and Methods for detailed description of
procedures).

relationship underscores the rejection of the hypothesis

that host range is primarily dependent on ability of

rhizobia to adsorb to host roots, at least for the

differential nodulation of Clark and its isoline, Clark-ril.

Clark (1957) reported that similar numbers of rhizobia

were recovered from the roots of plants carrying the rJlr11

genotype and plants that carried a nonrestrictive genotype

whether they were grown in a greenhouse or in the field.

Elkan (1962) demonstrated that Clark-ril actually maintained

substantially higher populations of rhizobia in rhizosphere

soil in the field than did Clark for 45 out of the first 60

d of plant growth. These observations, in conjunction with

the results of the present study, suggest that the limiting

step in nodulation of these plants occurs post-adsorption.

Pueppke (1984b) showed that adsorption of R. japonicum

strain 138 to roots of the soybean cultivar Hardee is

temperature sensitive; when that combination was subjected

to assay temperatures of 4 C, 27 C, and 37 C, the optimum

binding temperature was 27 C. The number of bacteria bound

per plant after co-incubation for 1 hr was reduced

approximately 90% at 4 C and approximately 65% at 37 C,

compared to the number bound at 27 C. In the present study,

the binding of strain 94 to roots of Clark-ril similarly was

temperature-sensitive. The reduction in the number of

bacteria bound per plant in 1 hr with a drop in assay

temperature from 27 C to 22 C was about 75%. The large

reduction in adsorption with only a 5 degree temperature

-75-

difference suggests that this combination is either more

highly temperature sensitive than the combination used in

the previous study (1984b), or that perhaps the curve of

temperature sensitivity for both combinations is very steep

on either side of an optimum temperature. The shape of the

response curve has not yet been determined, and would

require testing adsorption at considerably more temperatures

than have been used as treatments in either report.

For the combination of Clark-ril with strain 94, the

response of adsorption to temperature is opposite to that of

nodulation to temperature for the same (Tables 4.1 and 4.2

[data from Chapter Three], Devine and Breithaupt 1980b).

Although 40% of the plants were nodulated at 22 C, only 10%

were nodulated at 27 C. The number of bacteria bound per

plant after 2 hr at 22 C was 21 2. At 27 C 105 8

bacteria were bound, a five-fold increase. Clearly the

temperature effect on nodulation of these plants is

independent of its effect on binding.

The following conclusions are drawn from the data

presented in this report: i. Compared to 27 C, 22 C favors

nodulation in the Clark-ril x strain 94 combination,

whereas, the effect of temperature on adsorption is

precisely the opposite. ii. There is not a qualitative

difference between the adsorption of strains 94 and 110 to

Clark and Clark-ril soybean roots. iii. The rates of

adsorption in these combinations are similar to the rates

reported for other strain x soybean cultivar combinations

-76-

(Pueppke 1984b). iv. Under these experimental conditions,

there is no correlation between the number of rhizobia bound

to roots and the extent of nodulation of Clark or Clark-rji

soybean.

CHAPTER FIVE
INFECTION OF SOYBEAN ISOLINES DIFFERING AT THE RJ_ LOCUS
BY RHIZOBIUM JAPONICUM STRAINS WITH DIFFERENTIAL
NODULATING ABILITY

Figure 5.2. Clark soybean root hairs on a control
plant sampled and fixed 10 days after sham inoculation
with plant growth solution. The area sampled was the
10 cm of the primary root immediately above the point
representing the root tip at the initiation of the
experiment.